Prickly ash is listed in the pharmacopeia, with >30 prescriptions (Kono et al., 2020). The primary chemical constituents consist of alkaloids, amides, volatile, and fatty acids (Luo et al., 2022) with diverse functionalities. In African, Zanthoxylum species were used in traditional medicine for sickle cell anemia and malaria (Ouédraogo et al., 2019). In Japan and the United states, the Zanthoxylum-extract medicines are applied in studies on modern clinical trials (Rong et al., 2016). In Chinese, Zanthoxylum species were remedies of vomiting, ascariasis, itching, acute and chronic pain, and diarrhea (Zhang et al., 2021). Sanshools are polyunsaturated amides and are regarded as critical constituents in the biological activities of prickly ash. More than 10 sanshools have been identified since the first sanshool structure (α-sanshool) was reported (Crombie, 1952, 1954). The structure of sanshools was commonly subdivided into two parts, C-terminus and N-terminus (Sun et al., 2020). For example, there are 2–5 C-terminal unsaturated alkyl groups (Huang et al., 2012); unsaturated alkyls of the fatty chain could be oxidized to ketone, hydroxyl, or aldehyde groups. The N-terminal isobutyl could be dehydrogenated to isobutylene, or hydroxylated to 2′-hydroxy-isobutyl (Luo et al., 2022). In addition, differences in the position of oxidizing groups and the presence of cis and trans- double bonds can also generate isomers with distinct biological activities. N-isobutylamide sanshools incite burning oroxensation whereas hydroxylated congeners were reported to be anesthetic (numbing) orosensory recognition (Chruma et al., 2018). Therefore, whether there is difference among the abilities of antioxidant in sanshools with various structure?

Antioxidants are among the most studied topics both in nutrition and food science (Liu Y. et al., 2022; Liu R. et al., 2022). Antioxidants are compounds that can interact with free radicals and suppress chain reaction-related diseases (such as certain cancers, cardiovascular diseases, type 2 diabetes, chronic inflammation, aging, and the maintenance of gut health) (Rice-Evans and Diplock, 1993; Bartoszek and Polak, 2012). Although oxidative stress may be caused from enhanced levels of both reactive oxygen species (ROS) and reactive nitrogen species (RNS), researches on cell responses to sanshools have been focused on the influence of ROS to date. It has been reported (Brewer, 2011) that extracts of prickly ash have strong antioxidant activities that inhibited an overactive peroxidase system and scavenged free radicals in pathological models (Brewer, 2011; Falowo et al., 2014; Deng et al., 2019). These extracts reduced the production of malondialdehyde (MDA) in rat liver and protected primary Sprague Dawley rat hepatocytes against H₂O₂-induced cytotoxicity by inhibiting depletion of mitochondrial membrane potential and intracellular ROS generation (Zhang et al., 2017). In addition, hydroxy-α-sanshool (HAS) inhibited oxidative stress by reducing MDA production and increasing the activity of antioxidative enzymes (Li et al., 2020; Liu Y. et al., 2022; Liu R. et al., 2022). The above findings suggest that sanshools may activate a more comprehensive and even wider system of antioxidative responses to control redox balance. The nuclear transcription factor NF-E2-related factor 2 (Nrf2) is a helix–loop–helix basic leucine zipper transcription factor and controls the activation of downstream target genes coding for a network of enzymatic antioxidants. Under normal and balanced redox homeostasis, Kelch-like ECH-associated protein 1 (Keap1) binds to Nrf2 and promotes its degradation through the ubiquitin proteasome pathway (Kaspar et al., 2009). The dissociation of Nrf2 and Keap1 was promoted by the increased level of intracellular ROS during oxidizing conditions and Nrf2 escapes subsequent degradation (Ma, 2013). After that, the released Nrf2 is translocated to the nucleus, driving the expression of Nrf2 target genes (Ma, 2013).

Most studies focused on the capacity of scavenging free radicals and signaling pathways associated with antioxidant activity, but the relationship between sanshools with different structure and antioxidant ability was rarely reported and need to be illuminated (Luo et al., 2022). The abundant (>90%) components of sanshools in prickly ash are HAS, HBS, HRS, and RS, which vary in a hydroxyl group, N-alkyl group, and double bond (Figure 1A). Therefore, our objective was to explore the structure–activity relationships of sanshools on antioxidant activities in vitro and vivo. Antioxidant effects of four sanshools (HAS, HBS, HRS, and RS) against oxidative stress were assessed to elucidate structure–activity relationships. The study investigated the levels of oxygen radical absorbance capacity, radical scavenging capacity, mitochondrial membrane potential, intracellular reactive oxygen species, antioxidant activities, and expression of the signaling pathway caused by sanshool. We concluded that sanshools could act as substitutes for natural antioxidants, in which HRS has the highest antioxidant activity.

Sanshools and hydroxy-sanshools are the predominant class of long-chained polyunsaturated amides which are characterized by the presence of amide bond (−C(=O) NH−). These alkylamides are from the same family and are responsible for the tingling, numbing, and buzzing mouth sensation. Apart from utilization as an attractive flavoring, the favorable effects of prickly ash have been extensively documented and presented pre-clinic or clinical potentiality in human health promotion. Antioxidant activities have been attested in extracts of prickly ash based on the DPPH or other radical assays (Li et al., 2020). Except the Zanthoxylum genus, alkylamides also present in Brassicaceae, Convolvulaceae, Euphorbiaceae, Piperaceae, Poaceae, and Solanaceae. These compounds are consisted with saturated or unsaturated fatty acid chain and cyclic, aliphatic, or aromatic amine moiety, all possessing the antioxidant effects (Elufioye et al., 2020). Having that in mind, we aimed to elucidate the effect of HAS, HBS, HRS, and RS in eradicating free radicals, ameliorating intestinal injury and cytoprotective against H₂O₂-induced oxidative stress to further illustrate structure–activity relationships of sanshools.

To fully explore relationships between sanshool structure and antioxidant abilities, ORAC, DPPH, and ABTS assays were used to assess antioxidant abilities of all four sanshools. As expected, all sanshools have abilities of scavenging free radicals. The radical scavenging activity of sanshools is due to the hydrogen-donating ability and the presence of other H-donating groups (hydroxy and amide) (Xiao et al., 2022). Based on previous studies, the hydroxyl group is the main antioxidant site of many antioxidants, including polyphenols, phytic acid, catechin, phospholipids, and tocopherols (Son and Lewis, 2002). In this study, RS had the lowest antioxidant activity, due to its structure lacked the hydroxy which present in the other three sanshools. Antioxidative activity depends not only on the number of hydroxyl groups but also on the double-bond structure (Son and Lewis, 2002). Although double bonds involved in scavenging peroxyl radicals are contentious, antioxidant activities of HAS and HBS were lower than HRS which has more double bonds, implying that the double bond of sanshool was involved in the neutralization of peroxyl radicals. Similarly, it was also confirmed that cannabinoids with more double bonds than non-olivetolic had higher antioxidant activity (Dawidowicz et al., 2021). In addition, the antioxidant ability of HAS with cis-double bond was higher than HBS with trans-double bond. The previous study showed that substitution of a natural trans-double bond with a cis-double bond increased the oxidative ability of sphingomyelin by up to eight-fold, indicating that the trans-double bond is not essential for its antioxidant effects. Furthermore, a trans-double bond tends to be less reactive chemically than a cis-double bond, and the trans structure may also be less susceptible to free-radical reactions (Subbaiah et al., 2009). Perhaps this accounts for cis-isomer HAS with greater antioxidant activity than trans-isomer HBS, despite an identical molecular formula. Four sanshools prepared from the same species of prickly ash had different structures, which altered configurations and hydrogen-donating abilities (Cerdá et al., 2004; Mladenović et al., 2011). The IC50 value of HAS and HBS showed different effect on DPPḤand ABTS·scavenging activities, indicated that cis-isomer HAS exhibited more superior antioxidant activities on hydrosoluble free radical (ABTS·). Based on an assessment of antioxidant activities summarized above, we concluded that HRS had the best antioxidant activity, indicating it has the potential as a natural antioxidant and free radical scavenger. The above results confirmed that sanshool have the antioxidant ability, which were similar to previous studies (Liu et al., 2021; Peng et al., 2021). In another research, the sanshool possessed the synergistic antioxidant effects when it was mixed with other components (cichoric acid and caffeic acid derivative in Echinacea pallida) (Dalby-Brown et al., 2005). Relationships between the structure and antioxidant activities of sanshools are inherently complex, but our study provided novel insights.

Our results revealed that four sanshools (0–30 μM) have minimal toxicity on cell inhibition rate. Several studies showed that the sanshools extracted from leaves, fruits, bark, and roots of prickly ash exerted anti-proliferative effects. Extracts from the fruit inhibited cell proliferation in three human cancer cell lines (DLD-1, Caco-2, and HepG2) through JNK-dependent autophagic and no inhibition was observed in MCF-7, WiDr, and A549 (Nozaki et al., 2016). The growth of neurofibromatosis type 1 (NF-1)-deficient cancer xenografts in mice was blocked by the extracts of prickly ash through the PAK1/cyclin D1 pathway (Hirokawa et al., 2006). The 10 amides isolated from Nepalese Z. armatum were tested in a p53- and NF1-defective tumor cell line and only hydroxy-α-sanshool has no negatively impacting cell viability (Devkota et al., 2013). In our previous study, hydroxy-γ-sanshool inhibited cell proliferation of HCT-116 (IC50 = 88.01 μM) by activating P53 and Caspase 8 and had no cytotoxic effect to normal cells (IC50 = 481.52 μM). Therefore, the sanshools were not universally the key components which was used to inhibit the cell proliferation. In other word, the lower concentration of sanshools did not affect HCT-116 cell viability. In addition, H₂O₂ is widely used as an inducer of oxidative stress in vitro, generating hydroxyl radicals that cause serious damage to cells and attack biomolecules, ultimately leading to apoptosis or necrosis. In the present study, 200 μM H₂O₂ decreased cell viability and increased green fluorescence intensity in mitochondria of HCT-116 cells; however, treatment with sanshools reversed the decrease of cell viability and increased red fluorescence intensity. These results indicate that low-dose sanshools could protect HCT-116 cells from death induced by H₂O₂. Redox-active naphthoquinones linked with amide had superior activity in ATP rescue, which was maintained by the mitochondrial electron transport chain to increase the cell viability. Therefore, the protective effects of sanshools against mitochondrial dysfunction and cell death may have been due to the amide on the alkyl chain. Red fluorescence intensity in the RS group was lower than the other three sanshools, attributed to the hydroxyl group reducing oxygen entering the mitochondria and increasing ATP utilization or oxygen consumption, and attenuation of the formation of superoxide anions (Xiao et al., 2022). Although structures of HRS, HBS, and HAS differed, there was no significant difference in their protective effect on mitochondria membrane potential. Therefore, sanshools significantly reduced H₂O₂-induced cell death and mitochondrial dysfunction, with amide and hydroxyl groups implicated in maintaining mitochondrial membrane potential.

The increase in ROS level affects cell homeostasis, functions, and structures, resulting in oxidative stress. According to our findings, when HCT-116 cells were exposed to H₂O₂, there was an accumulation of intracellular ROS. However, this was improved with the administration of 30 μM sanshools. The protective effects of sanshools in the present research were partly mediated by ROS scavenging, given that sanshools treatment reduced the formation of MDA in colon tissues and cells, and changed the CAT activity of HCT-116. Similar results were obtained in previous research that the production of MDA in rat liver was reduced by the water extract of Szechuan pepper (Lu and Lu, 1995). Another recent research reported that HAS isolated from Zanthoxylum bungeanum regulates liver oxidative stress in hyperlipidemic rats by decreasing the levels of MDA (Li et al., 2020). Thus, due to their antioxidant abilities, direct action on ROS scavenging is one of the main protection mechanisms by sanshools. Accumulating evidence has demonstrated that there is an additional mechanism by which sanshools could reduce oxidative stress: indirect action on regulation of antioxidant enzyme. HBS was purified from Z. piperitum and can dose-dependently upregulates Gpx1 or Sod2 expression at 1.4–1.7 fold, protecting endogenous compounds from oxidative stress on mouse embryonic fibroblasts (Tomita et al., 2022). In the brain of D-gal/AlCl3 treated mice, the activities of SOD, CAT, and GSH-Px were improved by HAS at the doses of 2.5 and 5 mg/kg, while the MDA level was decreased (Liu Y. et al., 2022; Liu R. et al., 2022). SOD, CAT, and GSH-Px are three pivotal enzymes involved in ROS detoxification and suppress or prevent the production of free radicals (Ighodaro and Akinloye, 2018). Activities of these three key enzymes can be impaired and are usually accompanied by increased concentrations of malondialdehyde (MDA) (a marker for the degree of cellular oxidative damage) content under oxidative stress conditions (Nahar et al., 2016). In this study, all four sanshools significantly increased the activities of SOD, CAT, and GSH-Px in HCT-116 cells, similar to a previous report (Li et al., 2020). That HAS was superior to HBS in improving antioxidant enzyme activity was attributed to the spatial structure maintenance of the cis-double bond (Guerrero et al., 2012). In addition, the hydroxylation pattern on the core scaffold of compounds was reported to be closely associated with their affinity for antioxidant enzymes (Zhao et al., 2020) and that was also confirmed in natural flavonoids with abundant types (Zhang et al., 2019). Although RS induced the least improvement in antioxidant enzyme activities (CAT and GSH-Px) among the four sanshools, it also had the best inhibition of MDA production induced by H₂O₂. MDA was chemically reactive due to its carbonylic functions, whereas the potential OH group seemed not to react with carbonylic groups of MDA (Tsikas, 2017).

The accumulation of free radicals can disturb the physiological homeostasis. According to the studies, several signaling pathways could be rescued or accentuated by sanshool on the process of scavenging free radical, such as TLR4/NFκβ (Hong et al., 2017), MAPK (Wang et al., 2017), Nrf2 (Liu Y. et al., 2022; Liu R. et al., 2022), and PI3K/AKT (Li et al., 2020) signaling pathway. In our study, the Nrf2 pathway was activated in vitro and in vivo. Normally, Nrf2 is localized in the cytoplasm combined by Keap1. However, in the presence of oxidative stress, Nrf2 is released to the cell nucleus and activates many downstream antioxidant proteins such as heme oxygenase-1 (HO-1) to maintain the cell microenvironment homeostasis. In present western blot assay, all four sanshools had different protective effects against oxidant damage by activating the Keap1/Nrf2 signaling pathway, with the variation potentially due to several factors (Ma, 2013). Firstly, these four sanshools have different structures. Secondly, differences between HAS and HBS could be that the steric effect perturbs planarity and the ability to delocalize electrons. Thirdly, protein expression is regulated not only by the antioxidant abilities of four sanshools but also by the complex coordination of intracellular or in body microenvironments (Zhang et al., 2001). The protective of all four sanshools against oxidant damage was by activating the Keap1/Nrf2 pathway, with HRS having the most profound effects on the expression of proteins. Through molecular docking predictions, we found that there are various binding sites and several hydrogen bonds between Nrf2 and four sanshools. The results also showed that sanshool can effectively promote the shift of Nrf2 into the nucleus of the cell model, effectively alleviating oxidative stress. Erythritol bound with Nrf2 promoting a movement of Nrf2 protein into the nucleus to alleviate nonalcoholic fatty liver disease (Jin et al., 2021), although its binding energy (−2.55 kcal/mol) was inferior to the interaction between sanshools and Nrf2. The above results confirmed that sanshools have a strong binding effect with Nrf2; implying sanshools promote the activation of Nrf2. Conversely, different binding sites were formed by Nrf2 protein with sanshools, indicating differences among structures in binding energy and degree of Nrf2 activation, which affects the ability to mitigate H₂O₂-induced damage.

This study has confirmed the favorable effects of sanshools on efficiently inhibiting the oxidative stress. It has been shown that four sanshools scavenged ROS, increased the activity of antioxidant enzymes and decreased MDA concentration. Expression of Nrf2 protein and its downstream antioxidant protein HO-1 were positively increased, whereas Keap1 protein was decreased by sanshools. In particular, HRS had the best antioxidant activity, due to the number of cis-double bond, amido bond, and hydroxy group. To summarize, sanshools, especially HRS, had substantial antioxidant activities. This study provided new information regarding the structure–activity relationships of sanshools that may lead to new antioxidants.

The original contributions presented in the study are included in the article/supplementary materials, further inquiries can be directed to the corresponding author.

The animal study was approved by approval no. EAE-GZU-2022-E025. The study was conducted in accordance with the local legislation and institutional requirements.

ZC: Data curation, Formal analysis, Investigation, Methodology, Writing – original draft. LT: Validation. LL: Funding acquisition, Investigation. HW: Formal analysis, Methodology. XF: Funding acquisition, Validation. XL: Conceptualization, Funding acquisition, Project administration, Resources, Writing – reviewing & editing. CR: Funding acquisition, Resources.